Quantitation of Gene-Specific DNA Damage by Competitive PCR
| Autor: | Melihat Fidan, Philip Kurian, Lawrence P. Fernando, Daniel J. Fernandes |
|---|---|
| Rok vydání: | 2002 |
| Předmět: |
DNA damage
Genes myc Biophysics Antineoplastic Agents Apoptosis Biology Polymerase Chain Reaction Biochemistry DNA sequencing law.invention Necrosis Direct DNA damage chemistry.chemical_compound law Tumor Cells Cultured medicine Humans Molecular Biology Gene Polymerase chain reaction Teniposide Cisplatin Cell Biology Molecular biology chemistry Primer (molecular biology) DNA DNA Damage medicine.drug |
| Zdroj: | Analytical Biochemistry. 306:212-221 |
| ISSN: | 0003-2697 |
| DOI: | 10.1006/abio.2002.5705 |
| Popis: | A sensitive assay for quantitating DNA damage within individual genes would be a valuable tool for identifying the molecular mechanisms of disease and the sites of action of various carcinogens and anticancer drugs. This report describes a competitive PCR assay that was used to quantitate DNA damage induced by anticancer drugs within a 683-bp region of the c-myc gene in human CEM leukemia cells. Absolute quantitation of gene-specific DNA damage (attomoles or molecules of damaged DNA sequences) was achieved by coamplification of a homologous internal standard that has the same primer binding sites and PCR amplification efficiency as c-myc. The variability (standard error) associated with four separate determinations of the amount of c-myc sequence in 300 ng of DNA from untreated cells (6.80 +/- 0.05 SE amol) was less than 1% of the mean. The assay was capable of quantitating direct DNA damage that was induced by therapeutic concentrations of VM-26 and cisplatin prior to the onset of cellular apoptosis or necrosis. Both VM-26 (1-10 microM) and cisplatin (25-100 microM) induced a dose-dependent decrease in the amount of intact c-myc sequence. This assay should be readily adaptable to current real-time PCR protocols. |
| Databáze: | OpenAIRE |
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